Sulfate and thiosulfate transport in Escherichia coli K-12: nucleotide sequence and expression of the cysTWAM gene cluster.

The nucleotide sequence of the sulfate and thiosulfate transport gene cluster has been determined and located 3' to the gene (cysP) encoding the thiosulfate-binding protein. Four open reading frames, designated cysT, cysW, cysA, and cysM, have been identified. Similarities in primary structure were observed between (i) the deduced amino acid sequences of CysT and CysW with membrane-bound components of other binding protein-dependent transport systems, (ii) that of the CysA sequence with the "conserved" component of such systems, and (iii) that of the CysM sequence with O-acetylserine sulfhydrylase A (cysK gene product) and the beta-subunit of tryptophan synthase (coded by trpB). Expression of the four genes was analyzed in the T7 promoter-polymerase system.     

Sulfate and thiosulfate transport in Escherichia coli K-12: evidence for a functional overlapping of sulfate- and thiosulfate-binding proteins.

In Escherichia coli, sulfate and thiosulfate ions are transported by an ABC-type transporter consisting of both the membrane components (the products of cysT, cysW, and cysA genes) and the periplasmic binders (the products of cysP and sbp genes). The single cysP and sbp mutants are able to utilize both sulfate and thiosulfate as a sole sulfur source, while the inactivation of both genes leads to cysteine auxotrophy resulting from the block in the transport of both ions.     

Phosphate-specific transport system of Escherichia coli: nucleotide sequence and gene-polypeptide relationships.

The DNA nucleotide sequence of four genes for the phosphate-specific transport system of Escherichia coli is reported. Along with the DNA sequence for the phoS gene reported previously (Surin et al., J. Bacteriol. 157:772-778, 1984; Magota et al., J. Bacteriol. 157:909-917, 1984), this study completes the nucleotide sequence of the phosphate-specific transport region. The complete sequence (including phoS) contains five open reading frames oriented in the same direction, each preceded by a putative ribosome-binding site near the presumed translation initiation codon ATG. The complete sequence is transcribed counterclockwise, in the order phoS pstC pstA pstB phoU. Genetic complementation shows that of the four open reading frames in the new sequence, three correspond to known mutant alleles; the fourth, which was designated pstC, has not been described before and could not be related to any known mutant allele. We have confirmed that pstA was allelic to phoT32. The pstC, pstB, and phoU gene products were identified as peripheral membrane proteins. The pstA gene product appears to be an integral membrane protein.     

High-affinity L-arabinose transport operon. Nucleotide sequence and analysis of gene products

The nucleotide sequence of the "high-affinity" L-arabinose transport operon has been determined 3' from the regulatory region and found to contain three open reading frames designated araF, araG and araH. The first gene 3' to the regulatory region, araF, encodes the 23-residue signal peptide and the 306-residue mature form of the L-arabinose binding protein (33,200 Mr). The binding protein, which has been described elsewhere, is hydrophilic, soluble and found in the periplasm of Escherichia coli. This gene is followed by an intragenic space of 72 nucleotides, which contains a region of dyad symmetry 23 nucleotides long capable of forming an 11-member stem-loop. The second gene, designated araG, contains an open reading frame capable of encoding an equally hydrophilic protein containing 504 residues (55,000 Mr). Following a 14-nucleotide spacer, which does not appear to have any secondary structure, the third open reading frame, herein designated araH, is capable of encoding a hydrophobic protein containing 329 residues (34,000 Mr) that can only be envisioned as having an integral membrane location. 3' to araH there is a T-rich region containing a 24-nucleotide area of dyad symmetry centered 55 nucleotides from the termination codon. Analysis of the derived primary sequences of the araG and araH products indicates the nature and potential features of these components. The araG protein was found to possess internal homology between its amino and carboxyl-terminal halves, suggesting a common origin. The araG gene product has been shown to be homologous to the rbsA gene product, the hisP product, the ptsB product and the malK product, all of which presumably play similar roles in their respective transport systems. Putative ATP binding sites are observed within the regions of homology. The araH gene product has been shown to be homologous to the rbsC gene product, which is the first observed homology between two purported membrane proteins.     

Analysis of the entire nucleotide sequence of the cryptic plasmid of Chlamydia trachomatis serovar L1. Evidence for involvement in DNA replication.

Chlamydia trachomatis serovar L1/440/LN possesses a 7498bp plasmid which was designated pLGV440. The plasmid was cloned at the BamH1 site of pAT153 into Escherichia coli and the recombinant plasmid was designated pCTL1. A detailed restriction endonuclease map of pCTL1 was constructed. A fragment of the chlamydial plasmid was shown to function as a promoter in E. coli when placed upstream of the lacZ gene. The entire plasmid was sequenced by the chain termination method. Open reading frames were identified from the resulting consensus sequence together with a candidate for the plasmid origin of replication consisting of four perfect tandem repeats of a 22bp sequence, an A:T rich sequence and an open reading frame which could generate a 34.8kdal product. The predicted polypeptide products of the open reading frames were compared by computer with all reported protein sequences. Homology of the predicted polypeptide product of an open reading frame to the E. coli dnaB protein and the analogous product of gene 12 of bacteriophage P22 is described.     

Molecular characterization of a gene region involved in lipopolysaccharide biosynthesis in Bradyrhizobium japonicum: cloning, sequencing and expression of rfaf gene

A 3.0-kb region involved in lipopolysaccharide biosynthesis in Bradyrhizobium japonicum was sequenced. One complete open reading frame was identified which encodes a polypeptide of 354 amino acid residues with a predicted molecular mass of 38 209 Da. Expression of the protein using a T7 gene expression system revealed a band of similar molecular mass after sodium dodecyl sulfate polyacrylamide gel electrophoresis. A database search against known gene sequences revealed a significant sequence similarity to the rfaF gene cloned from several Gram-negative bacteria. The rfaF gene is known to encode heptosyltransferase II that transfers a second heptose to the inner core of lipopolysaccharide. The cloned B. japonicum open reading frame was able to functionally complement a rfaF mutant of Salmonella typhimurium SL3789. Transformation of this mutant with the B. japonicum gene restored production of an intact lipopolysaccharide and resistance to the hydrophobic antibiotic, novobiocin. An additional open reading frame having a significant sequence similarity to the rfaD gene was found to be divergently oriented to the rfaF gene.     

Molecular cloning and DNA sequence analysis of Escherichia coli topB, the gene encoding topoisomerase III

Escherichia coli contains two type 1 topoisomerases, topoisomerase I and III. Although topoisomerase III can be purified as a potent decatenase, its role in DNA metabolism is unclear. In order to address this issue, the gene encoding topoisomerase III from E. coli has been molecularly cloned and its DNA sequence determined. The cloned fragment of DNA contains an open reading frame that can encode a polypeptide of 73.2 kDa. The first 20 amino acids of this open reading frame are identical to those of topoisomerase III as determined by amino-terminal gas-phase microsequencing. Expression of the polypeptide encoded by this open reading frame, using a bacteriophage T7 transient expression system, results in the accumulation of a 74-kDa polypeptide. Soluble extracts prepared from cells overexpressing this gene product show a dramatic increase in topoisomerase activity when compared with control extracts. We propose that this gene be designated topB.     

Molecular cloning and sequencing of a gene from alkaliphilic Bacillus firmus OF4 that functionally complements an Escherichia coli strain carrying a deletion in the nhaA Na+/H+ antiporter gene.

A gene has been cloned from a DNA library from alkaliphilic Bacillus firmus OF4 that functionally complements a mutant strain of Escherichia coli, NM81, that carries a deletion for one of that strain's Na+/H+ antiporter genes (delta nhaA). The cloned alkaliphile gene restored to NM81 the ability to grow at pH 7.5 in the presence of 0.6 M NaCl and on 100 mM Li+ in the presence of melibiose, and concomitantly led to an increase in the membrane associated Na+/H+ antiport activity. The biologically active alkaliphile DNA was identified as an incomplete open reading frame, the sequence of which would encode a hydrophobic protein. The insert was used to isolate clones containing the complete open reading frame, which would be predicted to encode a protein with a molecular weight of 42,960 and multiple membrane spanning regions. When the open reading frame was expressed under the control of the T7 promoter, the gene product was localized in the membrane. Southern analysis indicated no homology between the alkaliphile gene, which we propose to call nhaC, and the nhaA gene of Escherichia coli, nor with other genes in digests of DNA from E. coli, Bacillus subtilis, or Bacillus alcalophilus. Although there was also no significant similarity between the deduced protein products of the alkaliphile gene and the nhaA gene of E. coli, there was a small region of significant similarity between the deduced alkaliphile gene product and the protein encoded by a human Na+/H+ antiporter gene (Sardet, C., Franchi, A., and Pouyssegur, J. (1989) Cell 56, 271-280).     

Nucleotide sequence of a gene cluster involved in entry of E colicins and single-stranded DNA of infecting filamentous bacteriophages into Escherichia coli.

Mutations in fii or tolA of the fii-tolA-tolB gene cluster at 17 min on the Escherichia coli map render cells tolerant to high concentrations of the E colicins and do not allow the DNA of infecting single-stranded filamentous bacteriophages to enter the bacterial cytoplasm. The nucleotide sequence of a 1,854-base-pair DNA fragment carrying the fii region was determined. This sequence predicts three open reading frames sequentially coding for proteins of 134, 230, and 142 amino acids, followed by the potential start of the tolA gene. Oligonucleotide mutagenesis of each open reading frame and maxicell analysis demonstrated that all open reading frames are expressed in vivo. Sequence analysis of mutant fii genes identified the 230-amino acid protein as the fii gene product. Chromosomal insertion mutations were constructed in each of the two remaining open reading frames. The phenotype resulting from an insertion of the chloramphenicol gene into the gene coding for the 142-amino acid protein is identical to that of mutations in fii and tolA. This gene is located between fii and tolA, and we propose the designation of tolQRA for this cluster in which tolQ is the former fii gene and tolR is the new open reading frame. The protein products of this gene cluster play an important role in the transport of large molecules such as the E colicins and filamentous phage DNA into the bacterium.     

Efficiencies of translation in three reading frames of unusual non-ORF sequences isolated from phage display.

An unusual nucleotide sequence, called H10, was previously isolated by biopanning with a random peptide library on filamentous phage. The sequence encoded a peptide that bound to the growth hormone binding protein. Despite the fact that the H10 sequence can be expressed in Escherichia coli as a fusion to the gene III minor coat protein of the M13 phage, the sequence contained two TGA stop codons in the zero frame. Several mutant derivatives of the H10 sequence carried not only a stop codon, but also showed frameshifts, either +1 or -1 in individual isolates, between the H10 start and the gene III sequences. In this work, we have subcloned the H10 sequence and three of its derivatives (one requiring a +1 reading frameshift for expression, one requiring a -1 reading frameshift, and one open reading frame) in gene fusions to a reporter beta-galactosidase gene. These sequences have been cloned in all three reading frames relative to the reporter. The non-open reading frame constructs gave (surprisingly) high expression of the reporter (10-40% of control vector expression levels) in two out of the three frames. A site-directed mutant of the TGA stop codon (to TTA) in the +1 shifter greatly reduced the frameshift and gave expression primarily in the zero frame. By contrast, a site-directed mutant of the TGA in the -1 shifter had little effect on the pattern of expression, and alteration of the first TGA (of two) in H10 itself paradoxically reduced expression by half. We believe these phenomena to reflect a translational recoding mechanism in which ribosomes switch reading frames or read past stop codons upon encountering a signal encoded in the nucleotide sequence of the mRNA, because both the open reading frame derivative (which has six nucleotide changes from parental H10) and the site-directed mutant of the +1 shifter, primarily expressed the reporter only in the zero frame.     

Molecular cloning and DNA sequencing of the Escherichia coli K-12 ald gene encoding aldehyde dehydrogenase.

The gene ald, encoding aldehyde dehydrogenase, has been cloned from a genomic library of Escherichia coli K-12 constructed with plasmid pBR322 by complementing an aldehyde dehydrogenase-deficient mutant. The ald region was sequenced, and a single open reading frame of 479 codons specifying the subunit of the aldehyde dehydrogenase enzyme complex was identified. Determination of the N-terminal amino acid sequence of the enzyme protein unambiguously established the identity and the start codon of the ald gene. Analysis of the 5'- and 3'-flanking sequences indicated that the ald gene is an operon. The deduced amino acid sequence of the ald gene displayed homology with sequences of several aldehyde dehydrogenases of eukaryotic origin but not with microbial glyceraldehyde-3-phosphate dehydrogenase.     

Characterization of a second gene involved in bacterio-opsin gene expression in a halophilic archaebacterium.

Southern blot analysis and nucleotide sequencing of DNA from three bacterio-opsin-deficient mutants of the archaebacterium Halobacterium halobium (M86, W105, and W109) revealed that they each contain an alteration in a region 2,000 to 3,800 base pairs (bp) upstream of the bacterio-opsin gene (bop). Nucleotide sequence analysis of this region, which is also located downstream of the previously characterized brp gene, revealed that it contains an open reading frame (ORF) of 2,022 bp. This 2,022-bp ORF has a start codon which overlaps the stop codon of the brp gene and is read in the same direction. The ORF could encode an acidic protein of 73,334 daltons (674 amino acids) with a predicted secondary structure typical of a soluble protein. Bop mutant M86 contains a 1,883-bp deletion extending from bp 351 of the ORF, to 197 bp beyond the stop codon. Mutant W105 has an ISH2 element integrated at bp 1239 of the ORF, and mutant W109 has an ISH26 element integrated at bp 1889. Our results suggest that the ORF is a gene (designated bat for bacterio-opsin activator gene) involved in bop gene expression.     

Cloning and characterization of the hemA region of the Bacillus subtilis chromosome.

A 3.8-kilobase DNA fragment from Bacillus subtilis containing the hemA gene has been cloned and sequenced. Four open reading frames were identified. The first is hemA, encoding a protein of 50.8 kilodaltons. The primary defect of a B. subtilis 5-aminolevulinic acid-requiring mutant was identified as a cysteine-to-tyrosine substitution in the HemA protein. The predicted amino acid sequence of the B. subtilis HemA protein showed 34% identity with the Escherichia coli HemA protein, which is known to code for the NAD(P)H:glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid synthesis. The B. subtilis HemA protein also complements the defect of an E. coli hemA mutant. The second open reading frame in the cloned fragment, called ORF2, codes for a protein of about 30 kilodaltons with unknown function. It is not the proposed hemB gene product porphobilinogen synthase. The third open reading frame is hemC, coding for porphobilinogen deaminase. The fourth open reading frame extends past the sequenced fragment and may be identical to hemD, coding for uroporphyrinogen III cosynthase. Analysis of deletion mutants of the hemA region suggests that (at least) hemA, ORF2, and hemC may be part of an operon.     

Nucleotide sequence of a staphylococcal plasmid gene, vgb, encoding a hydrolase inactivating the B components of virginiamycin-like antibiotics

The nucleotide sequence of a 1883 bp fragment isolated from a resistance plasmid harbored by a Staphylococcus aureus clinical isolate and carrying the gene, vgb, encoding a hydrolase inactivating the B components of virginiamycin family has been determined. The sequence contains one open reading frame which extends from the ATG codon at nt 641 to a TGA codon at nt 1537 and which potentially codes for a protein of 33.035 Da. This value is in agreement with the apparent size (33 kDa) of the protein observed, in minicell extracts. Inactivation of the B components of the virginiamycin antibiotics as well as resistance to these antibiotics were expressed in a virginiamycin sensitive mutant of Escherichia coli recipient containing the gene on a high copy number plasmid.